Treatment of intracellular bacterial infection

ABSTRACT

An intracellular bacterial infection in a plant or animal is treated by administration to a plant cell or animal cell of a particle to which an infectious bacteriophage is covalently attached, wherein the particle is internalised by the cell. Particles with phage attached and compositions comprising the particles are provided. A formulation, for treatment of a bacterial infection, comprises bacteriophage, liquid carrier and adhesive, which dries so that the adhesive adheres the bacteriophage to a surface, one such formulation comprising liquid carrier: 85%-99.98% by weight; bacteriophage: 0.01%-5% by weight; and adhesive: 0.01%-10% by weight.

FIELD OF THE INVENTION

The present invention relates to bacteriophage immobilised on a particlewherein the bacteriophage retains its infectivity. In particular, thepresent invention relates to treatment of bacterial infections inanimals and plants using those particles and to delivery of thoseparticles for such treatments.

BACKGROUND TO THE INVENTION

In recent years, as resistance to conventional antibiotics has continuedto grow and the application of chemical biocides becomes increasinglyunacceptable on environmental grounds, attention has turned toalternative methods for control of bacterial infection.

One promising approach involves the application of bacteriophages, beingnaturally occurring ubiquitous viruses that are harmless to humans,animals, plants and fish but lethal for bacteria. Bacteriophages arespecific and will infect only particular bacterial types, with severalsanitation products now on the market against pathogens such asSalmonella and Listeria.

Bacteriophage immobilised on a surface retain their infectivity and aremuch more resistant to degradation than free bacteriophage.Immobilisation to fine particulates, such as beads, allowsbacteriophages to be deployed by spray and aerosol and this mode ofdeployment has many applications, including treatment of human andanimal bacterial disease.

Pulmonary tuberculosis is the most predominantly occurring form oftuberculosis (Tuberculosis, 2005, 85, 227-234) and the currentchemotherapeutic regimen for treating pulmonary tuberculosis comprisesadministration of various antitubercular drugs such as isoniazid,rifampicin, ethambutol and/or pyrizinamide. Treatment is ineffective,leading to poor patient compliance and development of drug resistantstrains of the intracellular bacteria that cause the disease. WO2012/017405 provides an inhalable, microparticle based formulation.Still further therapies for this disease are, however, required.

Woiwode et al. (Chemistry and Biology, 2003, vol. 10, pp. 847-858)describes a phage display system that has been adapted to screensynthetic compounds. The synthetic compounds are attached to specificbacteriophage whose identity, and hence that of the synthetic compound,is encoded in the genome of the bacteriophage. A library of suchbacteriophage can be screened by conventional phage display techniquesand the identity of the synthetic compounds of interest can be found byidentifying the specific bacteriophage it is associated with.

Rizk at al. (Bioconjugate Chemistry, 2012, vol. 23, pp. 42-46) describesthe use of variant of substance P in receptor-mediated delivery of a‘cargo’ molecule across a cell membrane. Receptor mediated deliveryemploys the natural endocytosis of a ligand upon binding to itsreceptor. Substance P is an eleven amino acid neuropeptide ligand of theneurokinin type 1 receptor and can be linked to a suitable cargo via anon-reducible thioether bond. Thus suitable cargos bearing substance Pcan be endocytosed upon binding of substance P to its receptor. Suitablecargos include DNA fragments, polystyrene beads and M13 bacteriophage.

US 2009/0053789 describes a method of binding bacteriophage to particlesby exposing the particles to an electrical discharge in order toactivate them and then mixing the activated particles withbacteriophage. In this way the bacteriophage are covalently bound to theparticles.

US 2010/0285136 describes a system whereby bacteriophage are employed asa bridging molecule to bind particles comprising active agents to asubstrate. This is achieved by the bacteriophage bearing both a firstadditional peptide that adheres to the surface of the particle and asecond additional peptide that can adhere on substrate surfaces. Thissystem may be used for delayed release of active agents. A method ofscreening a combinatorial phage population to find the particularbacteriophage to use is also described.

WO 2008/109398 describes production of liposomes bearing modifiedbacteriophage for use in vaccine preparations. The vaccine antigen isdisplayed on a bacteriophage which is bound to the liposome.

US 2002/0001590 describes treatment of methicillin-resistantstaphylococcus aureus (MRSA) by exposing these pathogens tobacteriophage selected from the species Myoviridae. Formulationscontaining these bacteriophages and their use as bactericides are alsodescribed.

Broxmeyer (Medical Hypotheses, 2004, vol. 62, pp. 889-893) describes thetreatment an intracellular infection of macrophages using bacteriophageTM4 against virulent Mycobacterium tuberculosis and Mycobacterium avium.The bacteriophages were delivered to the pathogens as lysogens of thenon-pathogenic bacterium Mycobacterium smegmatis.

Lunov et al. (ACS Nano, 2011, vol. 5, pp. 1657-1669) describeinternalisation of carboxy and amino functionalised polystyrenenanoparticles by macrophages, and by differentiated and undifferentiatedcells from a monocytic cell line.

The present invention relates to novel deployment of bacteriophage fortreatment of bacterial infection.

An aim of the present invention is to provide compositions that areactive against the growth or persistence of bacteria present within thebody or cells of another organism, e.g. a plant or animal. An object ofparticular embodiments of the invention is to treat specific animal andplant intracellular infections.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a method of treatment orprevention of an intracellular bacterial infection in a plant or animal,comprising administration to a plant cell or animal cell of a particleto which an infectious bacteriophage is attached, wherein the particleis internalised by the cell.

In certain embodiments of the invention, an intracellular bacterialinfection in an animal can be treated or prevented by administration ofa particle of 1 micron or less in diameter to which an infectiousbacteriophage is attached.

Embodiments of the invention may be used to treat intracellularinfection in cells selected from epithelial cells, cells of theintestinal mucosa, polymorphonuclear cells, macrophages and monocytes.In specific examples described below in more detail, infection inmacrophages has been treated.

In certain embodiments of the invention, an intracellular bacterialinfection in a plant can be treated or prevented by administration of aparticle of 5 microns or less in diameter to which an infectiousbacteriophage is attached.

Also provided are a plurality of particles of mean diameter 5 microns orless, wherein infectious bacteriophage is attached thereto, for use intreating an intracellular bacterial infection in a plant, and aplurality of particles of mean diameter 1 micron or less, whereininfectious bacteriophage is attached thereto, for use in treating anintracellular infection in a plant or an animal. Still further providedare compositions comprising the particles.

It is preferred that the bacteriophage be covalently attached to theparticles.

DETAILS OF THE INVENTION

A method of treatment or prevention of an intracellular bacterialinfection in a plant or animal comprises administration to a plant cellor animal cell of a particle to which an infectious bacteriophage isattached, wherein the particle is internalised by the cell.

Following internalisation of the particle with phage attached, bacteriaresiding within the cell come into contact with and are infected andlysed by the phage, leading to phage progeny production within thebacterial cell and their subsequent release, leading in turn to furtherbacterial infection and lysis.

For treatment or prevention of an intracellular bacterial infection inan animal, a method of the invention may comprise administration of aparticle of 1 micron or less in diameter to which an infectiousbacteriophage is attached. The particles with phage attached may be 0.5microns or less in diameter and may suitably be 10 nanometres or more indiameter. In specific examples, described below in more detail,particles of approximately 100 nm diameter were taken up by humanmacrophages, demonstrating effective internalisation by cells to betreated.

WO 2003/093462 describes materials that the particles may be made from.For example, particles may be made from nylon and any other polymer withamino or carboxyl surface groups, cellulose or other hydroxyl-containingpolymer, polystyrene or other similar polymer, various plastics ormicrobeads including magnetic particles, or biological substances. Morepreferably, particles are made of a material commonly used intherapy/medicine; for example microbeads, which can be ingested orinhaled.

Delivery of the particles to animal cells may be via different routes.The particles may be suitably administered by inhalation, for examplefor infections in the lungs. The particles may be administered byinjection, e.g. in formulations comprising physiologically compatiblesaline.

For delivery to the lungs, formulations comprising bacteriophageattached to particles in aqueous solution are sufficiently stable fordelivery by nebulisation. In this regard, a number of types of knowndesigns of nebulisers (including adaptive aerosol delivery nebulisersand dosometric nebulisers) can be used to deliver formulations of theinvention. One type of high efficiency dosometric nebuliser is describedin WO 2004/045689 and WO 2004/045690. Other suitable nebulisers aredescribed in WO 2001/019437 and WO 2001/076762.

Formulations of the invention suitable for oral administration can bepresented as discrete units, such as capsules, caplets or tablets. Theseoral formulations can also comprise a solution or a suspension in anaqueous liquid or a non-aqueous liquid. The formulation can be anemulsion, such as an oil-in-water liquid emulsion or a water-in-oilliquid emulsion. The oils can be administered by adding the purified andsterilized liquids to a prepared enteral formula, which is then placedin the feeding tube of a patient who is unable to swallow.

Soft gel or soft gelatin capsules may be prepared, for example bydispersing the formulations of the invention in an appropriate vehicle(vegetable oils are commonly used) to form a high viscosity mixture.This mixture is then encapsulated with a gelatin based film usingtechnology and machinery known to those in the soft gel industry. Theunits so formed are then dried to a constant weight.

The invention also provides compositions and formulations comprising theparticles and biologically acceptable carriers, which may be preparedfrom a wide range of materials. Without being limited thereto, suchmaterials include diluents, binders and adhesives, lubricants,plasticizers, disintegrants, colorants, bulking substances, flavorings,sweeteners and miscellaneous materials such as buffers and adsorbents inorder to prepare a particular medicated composition.

It will be appreciated that the pharmacological activity of thecompositions of the invention can be demonstrated using standardpharmacological models that are known in the art. Furthermore, it willbe appreciated that the inventive compositions can be incorporated orencapsulated in a suitable polymer matrix or membrane for site-specificdelivery, or can be functionalized with specific targeting agentscapable of effecting site specific delivery. These techniques, as wellas other drug delivery techniques, are well known in the art.

Compositions, formulations and uses of the invention are suitable for awide range of intracellular bacteria that infect animal cells, includingbut not limited to Yersinia spp., Neisseria gonorrhoeae, Shigella spp.,Shigella flexneri, Listeria spp., Listeria monocytogenes, Salmonellaenterica, Salmonella enterica serovar Typhimurium, Legionellapneumophila, Coxiella bumettii, Francisella tularensis, Mycobacteriaspp., Mycobacterium tuberculosis, Chlamydia spp., Escherichia coli,Rickettsia spp., Brucella spp., Ehrlichia spp. and Burkholderia mallei.

Within an animal host cell, bacteria can reside in two differentlocations. Either bacteria can be localized to a vacuole which may bederived from a phagosome formed during engulfment of the bacteria, orbacteria may colonize the host-cell cytosol. A major advantage of theintracellular location may be access to host metabolites to supportbacterial multiplication in a relatively safe location and avoidance ofseveral potent host defence mechanisms.

For instance, some bacteria such as Yersinia and Neisseria gonorrhoeaeinvade specific types of epithelial cells and once internalised withinthe host cell may remain safe from therapeutic or immune attack,enclosed in an internal vacuole bounded by host cell membrane ordispersed in the cytoplasm. Some bacteria, such as Shigella species, areable to multiply within host cells. Listeria monocytogenes is ingestedwith food and invades cells of the intestinal mucosa. Examples ofbacteria able to multiply inside a vacuole include Salmonella entericaserovar Typhimurium, Legionella pneumophila, Coxiella burnettii,Francisella tularensis, Mycobacterium tuberculosis and obligateintracellular Chlamydia spp. Listeria monocytogenes, Shigella flexneri,enteroinvasive Escherichia coli and some Rickettsia species are able toenter and replicate in the cytosol of mammalian cells. Other bacteria,such as Mycobacterium tuberculosis, Brucella and Legionella live andgrow within phagocytic cells of the immune system (polymorphonuclearcells, macrophages or monocytes) and employ various intracellularsurvival strategies. Legionella pneumophila invades pulmonarymacrophages and causes pneumonia. In some cases bacteria need specificvirulence factors in order to recognize, invade and multiply withineukaryotic cells, but for most the intracellular phase is useful buttransient. The intracellular state may also contribute to bacterialdissemination within the host and, after evading the host defences, theycan be released into the environment or be directly transmitted toanother host organism.

In examples illustrating the invention, phagocytosis by and uptake ofparticles of the invention bearing infectious phage has been achieved,thus demonstrating that particles of the appropriate size can beinternalized and thus available for treatment of intracellularinfections. Accordingly, following the invention, infections comprisingbacteria located in different parts of animal cell compartments can nowbe treated.

For treatment or prevention of an intracellular bacterial infection in aplant, methods of the invention comprise administration of a particle of5 microns or less in diameter to which an infectious bacteriophage isattached. The particles may be of 1 micron or less in diameter, or of0.5 microns or less in diameter and may also be of 10 nanometers or morein diameter.

Delivery of the particles to plant cells may be via different routes.The particles may be suitably administered as an aerosol, for example byspraying onto leaves or other plant material. The particles may beadministered by injection, for example directly into a plant, such asinto the stem. In certain embodiments of the invention the particles areadministered to the roots. This can be achieved by spraying or wateringplant roots with compositions comprising the particles. In otherembodiments, the particles are introduced into the xylem or phloem, forexample by injection or being included in a water supply feeding thexylem or phloem.

Compositions, formulations and uses of the invention are suitable for awide range of intracellular bacteria that infect plant cells, includingbut not limited to bacteria of the genera Pseudomonas, Erwinia,Pectobacterium, Pantoea, Agrobacterium, Ralstonia, Burkholderia,Acidovorax, Xanthomonas, Clavibacter, Streptomyces, Xylella, Spiroplasmaor Phytoplasma, one or more or all of which can be responsible fornecrotic lesions on leaves, stems and fruits, internal discolorationsand decay, galls, scabs, cankers and soft rots. Trees, shrubs andherbaceous plants are all affected.

Losses from infection by the soft rot group of bacteria are especiallyimportant. They attack nearly all fruit and vegetables and can causedecay within hours. Losses of 5 to 10 percent are not uncommon.Organisms that cause soft rot live for long periods in the soil so thatinfection may occur before harvest. An embodiment of the inventioncomprises treating soil, e.g. applying a composition of the invention tosoil, prior to planting. Other embodiments comprise applying acomposition of the invention to soil and/or plant(s), optionally atleast one or more or a plurality of times during growth of the plant(s).

Copper compounds (such as Bordeaux mixture), although primarilyfungicidal, may have some effect. Antibiotics (streptomycin and/ortetracycline) may control bacteria but not cure already-diseased plants.Trees have been injected with tetracycline in the early stages ofinfections with some effect, but the cost and environmental concernsmake antibiotics impractical. The use of biological control has beensuggested; indeed the first suggestion of using bacteriophages tocontrol plant disease was made in 1937. Bacteriophages have been foundin association with buds, leaves, root nodules, roots, rotting fruit,seeds, stems and straw, crown gall tumours and in a wide range ofplants. Conventionally, spraying the plant is the method employed totreat leaf and stem surfaces, however the extent of internalisation isminimal. Therefore these methods are not suitable for treating bacterialinfections present beyond the accessible surface layers of a plant, orwithin the cells of a plant. The invention now provides for treatment ofintracellular bacteria infections of plants.

Compositions are provided by the invention for treatment or prevent ofbacterial infections in accordance with all aspects of the invention.Hence, the invention provides a plurality of particles of mean diameter5 microns or less, wherein infectious bacteriophage is attached thereto,for use in treating an intracellular bacterial infection in a plant oranimal. The plurality of particles may in embodiments have a meandiameter of 1 micron or less, especially for use against animal cellinfections. Particle size is suitably measured using methods andapparatus recognized as standardised in the art. Examples of sizingequipment are made by Malvern Instruments, using laser diffractionmethods.

In further embodiments of the invention, the plurality of particles havea mean diameter in the range 10 nm to 1 micron, suitably less than 1micron, suitably 50 nm or greater, suitably 800 nm or less.

In specific embodiments of the invention described in greater detailbelow, particles of the invention have a mean diameter of approximately100 nm.

In further preferred embodiments of the invention, substantially all ofthe plurality of particles have diameters less than 1 micron, morepreferably 90% or more have diameters less than 1 micron, and morepreferably 95% or more have diameters less than 1 micron.

Compositions are provided, comprising a plurality of these particles.The compositions may optionally also include a carrier, such as aliquid. In examples described in more detail below, compositions of theinvention comprise an aqueous carrier and bacteriophages immobilizedonto particles.

The carrier can also comprise an adhesive. In use a composition isapplied to a plant or animal and the particles are adhered to a surface(e.g. plant surface, leaf, stem or a surface of an animal, e.g. a bodypart, skin, organ surface) by the adhesive. In an aqueous carrier,adhesive is dissolved in solution, or may be suspended in an aqueouscarrier, and after application the composition dries, leaving a residueof adhesive that holds the particles in place. Further description ofuse of adhesives is set out below.

Further preferred embodiments of the invention comprise multiplebacteriophages wherein the bacteriophages are active against differentstrains of bacteria.

Particles for use in the invention to which bacteriophage areimmobilised, preferably by covalent bonding, are generally substantiallyinert to the plant or animal cell to be treated. In examples, nylonparticles (beads) were used. Other inert, preferably non-toxicbiocompatible material may be used. In addition, the particle may bemade of a biodegradable material.

It is further optional for the compositions to be adapted to increasetheir effectiveness. Particles can be modified to increase uptake and/orinternalisation of the particle by a plant or animal cell. For example,the particle can be functionalised by the addition of carboxyl groupsand/or functionalised by the addition of amino groups. One effect isthat particles may enter different compartments of the target eukaryoticcell. For example carboxy- and amino-functionalized polystyrenenanoparticles of approximately 100 nm in diameter have been shown to beinternalized by human macrophages and other cells by a diverse range ofmechanisms.

The particles can be treated so as to have a positive surface charge,for example by immobilisation of positively charged amino acids on theparticle surface.

In further embodiments of the invention, lectins which bind to bacterialtoxin or enzymes which degrade the toxin, or a combination of both, canbe co-immobilised with bacteriophages onto particles or onto separateparticles which are delivered at the same time. Pathogenic bacteria canexert their effects by various such toxins and the destruction ofbacteria, either by chemical or natural means (such as bacteriophages)can result in an increased, if transient, release of such toxins withthe resulting side effects being worse than the disease, and possiblyeven fatal. Use of lectins in this way can reduce this effect ofbacterial treatment.

Co-immobilisation of enzyme (to facilitate toxin degradation) withbacteriophage at the surface of a particle can also be used in theinvention. An enzyme suitable for modifying a product of bacterial lysiscan be attached to the particle.

Particles may comprise an opsonin, to improve phagocytosis. Examplesinclude complexes containing antibodies, in particular the Fc region,antigens and the C3 component of complement, that coat the surface ofbacteria. By co-immobilising bacteriophages at the surface of a particlewith opsonin-like components (e.g. combinations of antigen, antibody andC3 component of complement, or peptide or other fractions of each), incombination or individually, phagocytosis of the particle is promoted.

Iron is an essential nutrient for the growth and metabolism of nearlyall bacteria and an essential co-factor of numerous metabolic processes.In animal infection availability of iron is limited because iron issequestered by the high affinity binding proteins lactoferrin (mucosalsurfaces) and transferrin (serum). M. tuberculosis phagosomes containadditional transferrin receptors and upregulation may occur at membranesof other cell compartments containing intracellular pathogens. Inembodiments of the invention, the bacteriophage attached to the particleor the particle itself expresses or comprises transferrin orlactotransferrin. Such phage or particles may be may be preferentiallyphagocytosed by animal cells infected with intracellular bacteria and/orsubsumed within intracellular compartments containing bacteria. Inspecific embodiments of the invention, a bacteriophage is co-immobilisedonto a particle with transferrin or lactoferrin.

Additionally adhesion to a surface is enhanced in further embodiments ofthe invention in one or more of a number of ways.

-   -   The dispersing solution can be so composed that it provides an        adhesive function just sufficient to retain particles at the        desired site without interfering with the antibacterial action        of immobilised bacteriophage. This can be brought about by, say,        the addition of starch or other material.    -   The carrier particles can be charged to provide and promote        attachment to a surface perhaps having an opposite charge. This        can equally also apply to particles dispensed as powders or        within solution.    -   The carrier particles may have a ligand attached that promotes        attachment to a particular surface.

Thus, in a related invention, optionally for use in combination withother inventions and embodiments described elsewhere herein, there areprovided formulations with enhanced adhesion of bacteriophage tosurfaces to which the bacteriophage is applied. Hence, the relatedinvention provides adhering formulations, for treatment of a bacterialinfection, comprising bacteriophage, liquid carrier and adhesive. Inuse, the formulations dry so that the adhesive adheres the bacteriophageto a surface.

In such formulations, the carrier is suitably an aqueous carrier, andpreferably comprises or is water.

A typical formulation is composed mainly of the liquid carrier. Theproportions of the components will vary. In embodiments, theformulations comprises

-   -   liquid carrier: 85%-99.98% by weight;    -   bacteriophage: 0.01%-5% by weight; and    -   adhesive: 0.01%-10% by weight.

The liquid carrier by weight is preferably 90% or more by weight; thebacteriophage component (preferably in the form of particles withbacteriophage attached as described elsewhere herein) preferably makesup 0.1%-4% by weight; and the adhesive 0.1%-10% by weight, morepreferably 1% by weight to 5% by weight.

In use of formulations of the invention, bacteriophage-containingcompositions are applied by aerosol, and hence preferred formulationsare sprayable.

A method of treatment or prevention of bacterial infection comprisesapplying an adhering formulation of the invention to a surface andallowing the formulation to dry. Enhanced fixing of the bacteriophage,e.g. particles bearing the bacteriophage, is achieved, giving resistanceto loss of the bacteriophage and improved anti-bacterial activity insitu.

Particularly suitable adhesives are water-soluble, and examples ofadhesives for use in the invention include animal protein basedadhesive, plant-based glue, solvent-type glue, synthetic monomer glue,sugar, complex sugar, starch and a mixture of any of these.Specifically, the adhesive may consist of or comprise bone glue, fishglue, hide glue, hoof glue, rabbit skin glue, albumin glue, casein glue,meat glue, canada balsam (natural resin), coccoina, gum arabic (naturalresin), postage stamp gum, latex (natural rubber), library paste (astarch-based glue), methyl cellulose, mucilage, resorcinol resin,starch, urea-formaldehyde resin, polystyrene cement/butanone,acrylonitrile, cyanoacrylate (“superglue”, “krazy glue”), acrylicresorcinol glue, epoxy resin, epoxy putty, ethylene-vinyl acetate (ahot-melt glue), phenol formaldehyde resin, polyamide, polyester resin,polyethylene (a hot-melt glue), polypropylene, polysulfide,polyurethane, polyvinyl acetate, polyvinyl alcohol (PVA), polyvinylchloride (PVC), polyvinyl chloride emulsion, polyvinylpyrrolidone,rubber cement, silicones, styrene acrylic copolymer.

Examples of Specific Adhesive-Containing Embodiments of the Inventionare the Following Formulations:

Formulation A water: 92% by weight;  bacteriophage 3% by weight(covalently attached to particles of mean diameter 100 nm) PVA  5% byweight.

Formulation B water: 94% by weight;  bacteriophage 2% by weight(covalently attached to particles of mean diameter 100 nm) starch  4% byweight.

Bacteriophage for use in these adhering formulations are preferablyattached to substrates such as particles, for example as described in WO2003/093462 and WO 2007/072049. The bacteriophage are preferably also asdescribed herein for treatment of intracellular infections in plants andanimals.

The following optional and preferred features apply in relation to allinventions and embodiments thereof.

Immobilisation or attachment of bacteriophage to the particle substratemay be achieved in a number of ways. Preferably, bacteriophage areimmobilised via bonds, more preferably covalent bonds formed e.g.between the bacteriophage coat protein and the substrate.

Further, bacteriophage are preferably immobilised to the substrate viatheir head groups or nucleocapsid by activating the substrate before theaddition and coupling of bacteriophage.

The term “activated/activating/activation” is understood to mean theactivation of a substrate by reacting said substrate with variouschemical groups (leaving a surface chemistry able to bind viruses, suchas bacteriophage head or capsid groups).

Activation of said substrate may be achieved by, for example,preliminary hydrolysis with an acid, preferably HCl followed by a washstep of water and an alkali to remove the acid. Preferably, said alkaliis sodium bicarbonate. Binding of bacteriophage via their head groups isadvantageous. In the case of complex bacteriophage for example, bindingvia head groups leaves the tail groups, which are necessary forbacteria-specific recognition, free to infect, i.e., bind and penetratea host bacterial cell. A plurality of various strain-specificbacteriophage, may be immobilised to a substrate at any one time.

Coupling of phage to a substrate is as a result of the formation ofcovalent bonds between the viral coat protein and the substrate such asthrough an amino group on a peptide, for example a peptide bond.“Coupling Agents” that aid this process vary, and are dependent on thesubstrate used. For example, for coupling to the substrate nylon orother polymer with amino or carboxy surface groups the coupling agentscarbodiimide or glutaraldehyde may be used.

Further details of methods and preferred methods for attachment ofbacteriophage to particles are described in more detail in WO2003/093462 and WO 2007/072049, the contents of which are incorporatedby reference.

The invention is suitable for use with bacteriophage in general, withoutlimitation to the bacteriophage strain, though preferably with lyticbacteriophage.

Bacteriophage for the invention include bacteriophage in general withoutlimitation provided that the bacteriophage is obtainable and its host ortarget bacteria can be cultured and infected in culture. Thebacteriophage can be ssRNA, dsRNA, ssDNA or dsDNA bacteriophage, witheither circular or linear arrangement of the genetic material, and whichinfect cells of bacteria. The suitable bacteriophage include Myoviridae,Siphoviridae, Podoviridae, Lipothrixviridea, Rudiviridae,Ampullaviridae, Bacilloviridae, Bicaudaviridae, Clavaviridae,Corticoviridae, Cystoviridae, Fusseloviridae, Globuloviridae,Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae andTectiviridae.

The invention is now illustrated in specific embodiments with referenceto the accompanying drawings in which:

FIG. 1 shows a ×20 magnification micrograph of macrophages incubatedwith nylon beads carrying immobilised bacteriophages;

FIG. 2 shows a ×20 magnification micrograph of the result of anegative-control sample of the experiment shown in FIG. 1;

FIG. 3 shows a ×40 magnification micrograph of macrophages incubatedwith nylon beads carrying immobilised bacteriophages; and

FIG. 4 shows a ×40 magnification micrograph of the result of anegative-control sample of the experiment shown in FIG. 3.

EXAMPLE 1 Uptake of Submicron Polymeric Particles, with BacteriophageAttached, into Macrophages Experimental:

Nylon particles (100 nm mean diameter) containing immobilisedbacteriophages (phage Shield, host bacteria Salmonella typhimurium) wereincubated with CD14⁺ macrophages. The macrophages were visualised usinglight microscopy to determine the presence of beads and then washed andplated onto a lawn of host bacteria. Any surviving active immobilisedbacteriophages produce a “plaque” which highlights inhibition ofbacterial growth.

Cell Culture:

CD14^(÷) cells (macrophage) isolated from human blood samples werecultured in suspension in Dulbecco's Modified Eagle's Medium (DMEM)supplemented with 2 mM L-glutamine and 10% fetal bovine serum with a 5%CO₂ atmosphere at 37° C. A split ratio of 1:5 was used, the medium beingreplaced every 2 to 3 days.

Particle Production:

Nylon particles were treated by corona discharge (75 kV field) andrapidly added to a bacteriophage suspension at 1×10⁹ pfu/ml. Particleswere washed 3 times to remove non-bound bacteriophages.

Incubation:

Macrophages were seeded in tissue culture microscopy chambers at aconcentration of 5×10⁵ cells cm⁻² and cultured for 72 h at 37° C. Coronatreated beads were added at a concentration of 1×10⁹ beads/ml and thecells were incubated at 37° C. for 60 min. Cells were also inoculatedwith untreated 1×10⁹ beads and with no beads as a negative control.

Visualisation of Macrophages:

Macrophages were visualised using light microscopy at 20× and 40×magnification. Images of cells were taken using Cell-D microscopysoftware and the bead structures were measured using the softwaremeasurement grid to confirm the presence of beads inside the cell.

Results:

FIG. 1 shows a ×20 magnification micrograph of macrophages incubatedwith nylon beads carrying immobilised bacteriophages. The macrophageswere prepared by the method set out above and have internalised thebeads. Arrows indicate the phagocytosed particles. In FIG. 2 there is a×20 magnification micrograph of the result of the negative-controlsample of the experiment shown in FIG. 1. In this case the macrophageswere incubated in the absence of the nylon beads carrying immobilisedbacteriophages. In contrast to the result shown in FIG. 1, there is noevidence of phagocytosed particles.

FIG. 3 shows a ×40 magnification micrograph of macrophages incubatedwith nylon beads carrying immobilised bacteriophages. The macrophageswere prepared by the method set out above and have internalised thebeads. Arrows indicate the phagocytosed particles. In FIG. 4 there isshown a ×40 magnification micrograph of the result of thenegative-control sample of the experiment shown in FIG. 3. In this casethe macrophages were incubated in the absence of the nylon beadscarrying immobilised bacteriophages. In contrast to the result shown inFIG. 3, there is no evidence of phagocytosed particles.

EXAMPLE 2 Testing the Effect of Immobilised Bacteriophage on theInvasive Salmonella enterica Subsp Typhimurium Strain SL1344 inMacrophages Testing was Carried Out as Follows: Method

Raw 264 macrophage cells(http://www.lgcstandards-atcc.org/products/all/TIB-71.aspx?geo_country=gb)were seeded at 5×10⁴ cells/ml in a 24-well tissue culture plate in RPMI(RPMI-1640, Roswell Park Memorial Institute) medium containing glutamineand 10% fetal calf serum (FCS) and incubated overnight at 37° C., 5%CO₂.

The macrophage monolayers were activated overnight with 1 μg/mllipopolysaccharide (LPS) which was added to the media and incubatedovernight at 37° C., 5% CO₂. LPS-activated macrophages show a moreconsistent level of invasion by the Salmonella bacteria.

Macrophage monolayers were washed and new medium added withoutantibiotics. Various test combinations of immobilised bacteriophage andbacteria were added as detailed in the table 1, below.

TABLE 1 Test conditions. Combinations Time (hr) SL1344 alone 1 hrSL1344 + bacteriophage 1 hr SL1344 alone 2 hr SL1344 + bacteriophage 2hr SL1344 then bacteriophage 1 hr bacteria followed by another 1 hr withbacteriophage

100 μl of bacteria with or without bacteriophage were added to themacrophages. After the desired incubation time the medium was removedand the macrophages washed twice with 1 ml of phosphate-buffered saline(PBS). 1 ml of RPMI with gentamycin (100 μg/ml) was added and the cellsincubated for a further 1 hr (37° C., 5% CO₂). The medium was thenremoved and the monolayers washed twice with 1 ml PBS. The macrophageswere then lysed with 200 μl of 2% Triton X-100.

Samples were plated onto brain heart infusion (BHI) and BHI overlayscontaining SL1344 to enumerate the numbers of SL1344 and bacteriophages,respectively, that were internalised by the macrophages.

20 μl of sample dilutions were inoculated onto plates to deduce thenumbers of bacteria which were added onto the macrophage monolayers. Thenumbers of bacteriophage were estimated by adding 100 μl ofbacteriophage sample to 100 μl of overnight SL1344 culture in a 5 mlagar overlay.

SL1344 was added to each well at 3.5×10⁵ colony forming units per well.

Beads bearing immobilised bacteriophage—an estimated 1×10¹³ beads/mlwere diluted 1:100 and 100 μl added to each well. Thus it is estimatedthat 1×10¹⁰ beads/ml were added to each well. The number of SL1344invading the macrophages was then calculated.

Initial results showed in some cases elimination of infectious bacteriaand in others a reduction in the infectious load of bacteria in themacrophages. Further testing to quantify the results is ongoing.

The invention thus provides a method of treatment or prevention of anintracellular bacterial infection in a plant or animal and compositionssuitable therefor.

REFERENCES

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1. A method of treatment or prevention of an intracellular bacterialinfection in a plant or animal, comprising administering to a plant cellor animal cell of a particle to which an infectious bacteriophage iscovalently attached, wherein the particle is internalized by the cell.2. The method of claim 1, wherein the particle is 1 micron or less in adiameter.
 3. The method of claim 2, wherein the particle is 0.5 micronsor less in diameter.
 4. The method of claim 2, wherein the particle is10 nanometers or more in diameter.
 5. The method of claim 1, wherein theparticle is administered by inhalation or injection.
 6. The method ofclaim 2, wherein the bacteriophage infects Yersinia spp., Neisseriagonorrhoeae, Shigella spp., Listeria spp., Salmonella enterica,Salmonella enterica serovar Typhimurium, Legionella pneumophila,Coxiella burnettii, Francisella tularensis, Mycobacteria spp., Chlamydiaspp., Escherichia coli, Rickettsia spp., Brucella spp., Ehrlichia spp.or Burkholderia mallei.
 7. (canceled)
 8. A method of treatment of anintracellular bacterial infection in a human macrophage, comprisingadministering to the macrophage a particle of diameter 1 micron or lessto which an infectious bacteriophage is covalently attached.
 9. Themethod of claim 1, comprising administering to the plant a particle of 5microns or less in diameter to which an infectious bacteriophage iscovalently attached.
 10. The method of claim 9, wherein the particle is1 micron or less in diameter.
 11. The method of claim 10, wherein theparticle is 0.5 microns or less in diameter.
 12. (canceled)
 13. Themethod of claim 9, wherein the particle is administered as an aerosol.14. (canceled)
 15. The method of claim 9, wherein the particle isadministered to the roots.
 16. The method of claim 9, wherein theparticle is administered through being introduced into the xylem orphloem.
 17. The method of claim 9, wherein the bacteriophage infectsbacteria of the genera Pseudomonas, Erwinia, Pectobacterium, Pantoea,Agrobacterium, Ralstonia, Burkholderia, Acidovorax, Xanthomonas,Clavibacter, Streptomyces, Xylella, Spiroplasma or Phytoplasma. 18-20.(canceled)
 21. A composition comprising a plurality of particles of meandiameter 1 microns or less, wherein one or more infectiousbacteriophages is covalently attached thereto. 22-23. (canceled)
 24. Thecomposition of claim 21, comprising multiple bacteriophages, wherein thebacteriophages are active against different strains of bacteria.
 25. Thecomposition of claim 21, wherein the particle is made of a biodegradablematerial.
 26. The composition of claim 21, wherein the particles aremodified to increase uptake and/or internalization of the particle by aplant or animal cell.
 27. The composition of claim 21, wherein theparticle is functionalized by the addition of carboxyl groups. 28-43.(canceled)
 44. The method of claim 6, wherein the bacteriophage infectsShigella flexneri, Listeria monocytogenes, or Mycobacteriumtuberculosis.